Bridged Hybrid Monolithic Column Coupled to High-Resolution Mass Spectrometry for Top-Down Proteomics

Abstract

Top-down mass spectrometry (MS)-based proteomics has become a powerful tool for comprehensive characterization of intact proteins. However, because of the high complexity of the proteome, highly effective separation of intact proteins from complex mixtures prior to MS analysis remains challenging. Monolithic columns have shown great promise for intact protein separation due to their high permeability, low backpressure, and fast mass transfer. Herein, for the first time, we developed bridged hybrid bis(triethoxysilyl)ethylene (BTSEY) monolith with C8 functional groups (C8@BTSEY) for highly effective protein separation and coupled it to highresolution MS for identification of intact proteins from complex protein mixtures. We have optimized mobile phase conditions of our monolith-based reverse-phase chromatography (RPC) for online liquid chromatography (LC)−MS analysis and evaluated separation reproducibility of the C8@BTSEY column. We further assessed the chromatographic performance of this column by separating a complex protein mixture extracted from swine heart tissue. Using our monolithic column (i.d. 100 μm × 35 cm), we separated over 300 proteoforms (up to 104 kDa) from 360 ng of protein mixture in an 80 min one-dimensional (1D) LC run. The highly effective separation and recovery of intact proteins from this monolithic column allowed unambiguous identification of ∼100 proteoforms including a large protein, αactinin2 (103.77 kDa), by online 1D LC−MS/MS analysis for the first time. As demonstrated, this C8@BTSEY column is reproducible and effective in separation of intact proteins, which shows high promise for top-down proteomics.

Proteomes Are of Proteoforms: Embracing the Complexity

“…The exact m / z of the intact proteoforms of interest are then measured [ 142 , 143 , 144 ]. While this method removes any inference associated with bottom-up peptide MS and provides full sequence coverage of proteoforms, including number, position, and type of PTM on a single polypeptide chain, there remain many technical challenges to be addressed [ 87 , 145 , 146 ]. First, intact protein MS cannot match the high throughput obtained by peptide MS and is currently unable to handle large-scale analyses [ 143 , 147 ].…”

“…Currently, MS technologies are incapable of handling intact proteoforms of larger sizes and mixtures of proteoforms with different physico-chemical properties; this results in loss of certain components or incompatibility of the protein species with MS [ 78 ]. Thus, this method can only be consistently applied to species of less than ~30 kDa; there are some reports of select identifications of proteoforms >30 kDa, even up to 104 kDa, although this is completely dependent on the capacity of a few random species to effectively fragment using available technology [ 18 , 143 , 144 , 145 , 147 , 148 ]. Thus, in studies identifying larger proteoforms (e.g., >100 kDa), these are currently exceptions—the majority of species that can be effectively analyzed are in the low MW range (i.e., 3–30 kDa) and only a very small handful of larger MW species can currently be effectively identified.…”

“…As the MS-intensive approach can currently analyze primarily a lower MW sub-set of species, it is not yet capable of large-scale investigations of ‘complete’ proteomes [ 18 , 143 , 144 , 145 , 147 , 148 ]. In saying that, this method can comprehensively characterize proteoforms, strongly suggesting that MS-intensive top-down will be an extremely powerful tool in the future, when current technical limitations are overcome, enabling it to be more broadly applicable, routine, and far more cost-effective.…”

Capillary zone electrophoresis-tandem mass spectrometry with ultraviolet photodissociation (213 nm) for large-scale top–down proteomics

“…[7][8] Thousands of proteoforms and hundreds of proteins can now be identified and characterized using advanced liquid-phase separation methods, high-resolution mass spectrometers, and various gas-phase fragmentation techniques . [9][10][11][12][13][14] Traditional reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS) has been improved for top-down proteomics with the use of shorter bonded stationary phases (C2-C4) and longer columns. 15 Capillary zone electrophoresis (CZE) offers an alternative and high-capacity separation of proteoforms based on their sizes and charges.…”

Challenges and Advances in the Fabrication of Monolithic Bioseparation Materials and their Applications in Proteomics Research

“…Particularly, fast separation of seven proteins was realized within 2.5 min in cLC. More recently, Zhang's group fabricated a hybrid C8‐modified monolith via sol–gel process of bis(triethoxysilyl)‐ethylene for protein separation in cLC. The separation of over 300 proteoforms from 360 ng of proteins was achieved using a 35 cm long monolith, and about 100 proteoforms could be recognized by LC–MS/MS, demonstrating promise of the hybrid monolithic column for top‐down proteomics.…”